Mass spectrometry relates to the determination of the molecular weights of individual molecules by their conversion into ions in vacuo and then subjecting the ions to electric and/or magnetic fields to determine their mass. Ion formation is a prerequisite to the determination of a molecule's molecular weight by mass spectrometry.
Classical ionization methods involve gas phase interactions of the molecule to be ionized with electrons, as in electron impact ionization (EI), photons as in photo ionization (PI), and other ions as in chemical ionization (CI). These ionization methods result in the formation of ions from the neutral molecule by a variety of mechanisms, including the removal from or addition of an electron or a positively charged entity (e.g., a proton) to the molecule. While these classical ionization methods work well for relatively low molecular weight molecules that can be vaporized in vacuo, the extension of these methods to the analysis of large polar molecules, including large organic molecules, such as biopolymers, suffers from the difficulty associated with transforming these molecules into ions. Generally, large polar molecules cannot be vaporized without extensive decomposition.
The deficiencies of classical ionization methods for determining the molecular weight of biologically important molecules have resulted in the development of additional ionization methods directed to producing intact ions from molecules of increasing size. Several of these methods are based on the rapid deposition of energy to a surface upon which the molecule to be analyzed has been deposited. Rapid heating methods include plasma desorption (PD) and secondary ionization mass spectrometry (SIMS), also referred to as fast ion bombardment (FIB), in which the molecule deposited upon a surface is bombarded by ions (e.g., cesium ions) accelerated to energies in the tens of kilovolts. Fast atom bombardment (FAB), in which accelerated ions are neutralized prior to striking the surface, and laser desorption (LD), which involves the use of high energy photons to vaporize the molecule, are also included among these high energy techniques. These techniques have successfully produced intact ions from relatively large bio-organic compounds having molecular weights up to about 30,000 Daltons.
Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is a currently popular laser desorption method that has been refined to be particularly useful for mass analysis of high molecular weight biological molecules, such as peptides and proteins. In the method, a protein sample embedded in a light-absorbing matrix, made from a strongly ultraviolet or infrared light absorbing material, is irradiated by intense, short-duration pulses of laser light. The laser light results in the ablation of bulk portions of the protein-containing matrix and the formation of gas phase intact protein ions, the molecular masses of which can then be determined by mass analysis.
Advantages of the MALDI-MS method relate to the fact that biological samples can be examined without extensive purification, in the presence of other proteins, and can include common biochemical additives that do not interfere with the method; most classes of proteins can be examined provided that the protein can be dissolved in appropriate solvents; the total amount of protein required for analysis is in the range of from about 1-10 pmol; and perhaps most significantly, proteins having masses ranging to greater than 100 kDa can be analyzed. Typically, this ionization technique employs a time-of-flight (TOF) mass analyzer, which determines ion mass as a function of the time required for the ion to travel to the analyzer's detector. Thus, unlike other conventional mass analyzers, TOF mass analyzers do not have an upper nominal mass detection limit and are therefore particularly useful in determining the mass of high molecular weight ions.
The MALDI method is not without its limitations. Sample preparation is crucial to matrix-assisted laser desorption ion formation. The surroundings of the protein to be analyzed (i.e., the matrix) must be fashioned so that an intense light pulse can transfer the intact molecule into the gas phase. The matrix is generally a crystal into which the protein is incorporated. However, few compounds can form crystals that incorporate proteins, absorb light energy, and eject and ionize the protein intact. Furthermore, the formation of the protein-containing matrix is not a trivial process that reliably provides useful mass spectra. Although several matrix compounds are widely used, the selection of a matrix for a particular protein is empirical.
In contrast to mass spectrometric techniques that permit the continuous acquisition of mass spectral data from separation devices (e.g., chromatographs) that introduce sample to the ion source, MALDI is a "batch" method requiring substantial sample preparation for each analysis performed. Subtle variations of experimental parameters, for example, the matrix, matrix solvent, laser power, number of laser shots, presence of calibrant, and analyte-to-matrix ratio can cause dramatic changes in the outcome of the analysis. Thus, despite its qualitative analytical benefits, the method does not lend itself to quantitative mass analysis. MALDI-MS of proteins has been recently reviewed by Beavis and Chait in Methods in Enzymology, Vol. 270, 1996, pp. 519-551.
Other desorption ionization techniques employ strong electrostatic fields to desorb ions. The methods include thermospray (TS), atmospheric pressure ion evaporation (APIE), atmospheric pressure chemical ionization (APCI), and electrospray (ES) ionization, and generally involve ion desorption from small charged droplets of solution into a bath gas, which is subsequently admitted into the vacuum system of a mass analyzer. Of these techniques, electrospray has evolved into a powerful and widely practiced tool for the analysis of high molecular weight biological molecules. The success of ES in the analysis of biomolecules lies in the method's ability to extract fragile chemical species intact from solution, ionize them, and transfer them to the gas phase for mass analysis. A unique characteristic of the ES ion source is the ability to form multiply-charged ions, which facilitates the analysis of extremely high molecular weight molecules with mass analyzers having relatively low nominal upper mass limits. Electrospray ionization methods have been extensively reviewed. See, for example, reviews by Banks, Jr. and Whitehouse in Methods in Enzymology, Vol. 270, 1996, pp. 486-519; and Edmonds and Smith in Methods in Enzymology, Vol. 270, 1990, pp. 412-431.
In an ES ion source, a liquid sample is introduced through a small bore tube that is maintained as several kilovolts at or near atmospheric pressure into a chamber containing a bath gas. A strong electrostatic field at the tube's tip charges the surface of the emerging liquid generating coulomb forces sufficient to overcome the liquid's surface tension and to disperse the liquid into a fine spray of charged droplets.
In the ES ionization technique, an external electric field is employed for purposes of both the creation of a spray of fine droplets and for the formation of gas phase ions. The success of the ES ionization method is highly dependent upon the electrostatic field at the tip of the tube as well as other parameters. For example, if the field at the tip is too high, or the pressure of the bath gas too low, a corona discharge will occur at the tip and substantially decrease the effectiveness of the nebulization.
Despite the advances in ion formation achieved by ES ionization methods, the ES technique is not without limitation. A common problem encountered with low flow rate liquid chromatography/mass spectrometric (LC/MS) or infusion type atmospheric pressure ionization (API) inlet designs is unstable operation in negative ion mode. The problem is especially true for analyzing samples in aqueous solution. The problem is manifested in the mass spectra with the appearance of [H.sub.2 O].sub.n peaks and other noncovalent adducts. These artifacts are symptomatic of corona discharge, a common occurrence at nanoliter flow rates, where the more obvious indications of discharge seen at higher flows, such as excessively high electrospray current and disruption of the normal baseline, are often missing. Accordingly, there exists a need for an ionization method that affords the advantages associated with ES ionization, permits negative ion analysis free from adduct formation, and further provides stable ion currents with nanoliter flow rates.
Optimization of negative ion ES ionization, including ion current stability, for biological samples in aqueous solutions is often problematic. While the common practice of using oxygen or sulfur hexafluoride as electron scavengers at the spray tip is known to inhibit corona discharge, discharge problems often remain. Other sources of ion beam instability that are not affected by the presence of scavenger gas, also impact operation in negative ion mode. While efforts to optimize ES ionization using small interior diameter stainless steel capillaries worked extremely well for positive ion formation and detection, such efforts were less successful for negative ion mode. The result suggests that stainless steel has problems with signal stability at low flows with negative ions, especially in aqueous solutions with less than 20% or so organic solvent content.
In addition, ES negative ion experiments with hydrophobic glycolipids (e.g., lipid A) demonstrated that detection limits for the glycolipids, dissolved in chloroform/methanol solution where adduction problems are less severe due to the electron scavenging properties of chloroform and the relatively lower electrospray voltage required to produce useful mass spectra, were still poor compared to those routinely achieved with many positive ion protein and peptide applications. Flow rates below about 500 nL/min are also a problem with ES ion sources. Furthermore, clogging problems with small orifice (about 5 .mu.m inner diameter) nanospray tips are more severe than for peptide samples. However, because fused silica, a commonly used alternative to stainless steel capillaries, is a poor conductor of electricity, simply switching back to doing ES ionization with small inner diameter fused silica capillary tubes is not an attractive option. Accordingly, a need exists for an improved, highly sensitive method of forming negative ions using low sample flow rates that allow the greatest possible signal-to-noise (S/N) ratio for a given concentration, and maximizes resistance to capillary clogging during nanoliter scale infusion for the analysis of trace quantities of bacterial glycolipids.
As noted above, API methods that employ electrospray and atmospheric pressure chemical ionization sources have found widespread application in biology and chemistry. These devices allow gas phase ions to be formed from highly involatile and sensitive, delicate molecules. In standard ES ionization methods, charging, ionization and solvent evaporation all occur in or near a very small region commonly referred to as the Taylor cone. In order to properly form the Taylor cone and effectively perform ES ionization, flow rate, voltage, pressure, temperature, and solvent properties all have to be optimized over a relatively narrow range. As a result, ES ionization methods typically have a relatively limited range of applications. Accordingly, there exists a need for ionization methods and devices that overcome the deficiencies associated with standard ES ionization methods. More specifically, a need exists for methods and devices in which solution charging and spray formation can be independently optimized. A need also exists for ionization methods and devices having no externally applied high voltage and no strong electric field at the spray tip to avoid the problems associated with corona discharge. The present invention seeks to fulfill these needs and provides further related advantages.